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Overall Hypothesis IF N-glycans on PRRs are the first to recognize invading pathogens, THEN mutations in genes that encode for N-glycosylation enzymes will cause a decreased or non-existent immune response. Results: Figure 1 • Figure 1A & 1B Hypothesis: IF N-glycans recognize invading pathogens and stimulate a seedling growth arrest immune response, THEN mutations in genes that encode for N-glycosylation enzymes will leave growth unaffected. Figure 1 Background • Method: – tDNA insertion mutants – 100nM elf18 or flg22 treatment GOI Ti Plasmid Agrobacterium GOI - Gene of Interest ARM - Antibiotic Resistance Marker Figure 1A Out of ALL mutants, only stt3a-2 was strongly insensitive to MAMP treatment Results: Figure 1 • Hypothesis Supplemental Figure 2 IF N-glycans recognize invading pathogens and stimulate an “oxidative burst” immune response, THEN mutations in genes that encode for N-glycosylation enzymes will decrease the “oxidative burst” immune response. Results: Supplemental Figure 2 Figure 1B Background • Method: – Col-O and mutants treated with 0.5x108 cfu/ml of Pseudomonas psyringae pv. tomato DC3000 bacteria. • Hypothesis Figure 1B IF an immune response decreases bacterial viability, THEN mutations in N-glycosylation that decrease immune response will have no effect on bacterial viability. Figure 1B •Mutants showed to be more susceptible to bacteria Figure 2 Background • Method: – Cross-linking – SDS-PAGE • Hypothesis Figure 2A: IF peptide shape is essential to pathogen recognition, THEN cross-linked peptides will result in a loss of function for N-glycosylation mutants. Cross-linking • Radioactivity-labeled elf26 and flg22 peptides(MAMP variants) – in vitro – Bind to receptors EFR and FLS2 – If receptor is still present we will see a band at 150kDa (EFR) or 175kDa (FLS2) – Shows ligand binding and response SDS-Page • Separates proteins according to their size Figure 2A Results: Figure 2 • Hypothesis Figure 2B IF N-glycosylation is responsible for protein folding, then mutation in the N-glycosylation pathway will result in decreased PRR accumulation. Figure 2B Results: Figure 2 • Localization of PRRs in selected Nglycosylation mutants IF EFR and FLS2 are truly membrane-bound proteins, THEN a fluorescent tag on these PRRs will result in localization at the plasma membrane. Confocal Microscopy http://www.olympusfluoview.com/theory/index.html Figure 2C Supplemental Figure 5A & B Results: Figure 3 • Hypothesis for Figure 3 IF tunicamycin causes N-glycan degradation, THEN a gel will reveal band shift proportional to N-glycans present on wildtype PRRs. Figure 3A Figure 3B Figure 3C Figure 3D Results Figure 4 • Hypothesis for Figure 4A IF EFR function is solely based on NGlycosylation, THEN point mutations to elimate N-Glycosylation motifs will result in EFR dysfunction. EFR Figure 4A Figure 4B Figure 4C